Occurrence , distribution and dynamics of virus antigen accumulation in pepper cultivation on open fields in Republic of Macedonia during 2008-2009

Research was conducted in 2008 and 2009 in order to determine the virus status of pepper (Capsicum annuum) cultivated in open fields in the Republic of Macedonia (R. Macedonia). Most of the tested seedlings were virus free, but a few seedlings were found to be infected with Potato virus Y (PVY). During the vegetative season, in the tested areas pepper plants were mostly infected by Cucumber Mosaic Virus (CMV), while the infections by Alfalfa mosaic Virus (AMV), Tobacco Mosaic Virus (TMV), TSWV Tomato Spotted Wilt Virus (TSWV) and PVY were less present. None of the samples were infected with Potato virus X (PVX). With the performed analysis, dynamics of virus antigen accumulation during the production period was established, which was related with less visible symptom in some of the observed infected plants.


INTRODUCTION
The pepper is one of the most significant vegetable crops that are cultivated in Republic of Macedonia (R. Macedonia).During 2003, an example of a typical annual production, 7500 ha of pepper plants were cultivated in greenhouses and 843 ha in open fields, and the yields obtained were 19.56 tonnes per t/ha and 3.91 t/ha respectively.These yields do not fulfill the requirements for the domestic market, and the processing industry which has a tremendous growth potential (Ministry of Agriculture, forestry and water economy -facts and figures, 2003 loc.cit.Rusevski and Bandzo, 2004).The main problems associated with pepper production in R. Macedonia are virus infections (Jovancev et al., 1996).The virus infections are most commonly caused by Cucumber Mosaic Virus (TMV), Tomato Spotted Wilt Virus (TSWV), Potato virus X (CMV), Alfalfa mosaic Virus (AMV), Tobacco Mosaic Virus (PVX) and Potato virus Y (PVY) etc. (Jovancev et al., 1996).These pathogens can cause severe damages.Šutić (1995) notes that in the territory of the former Yugoslavia yield losses caused by CMV, AMV, TMV and other viruses on pepper ranged from 34 to 100%.In R. Macedonia the yield losses caused by different viruses on pepper varied from 10 to 100% (Jovancev et al., 1996).
In   (Bogatzevska et al., 2007).During 2009 the sampling was made from seedlings and grown pepper plants, from different varieties, such as: Kurtovska kapija, Palanechko chudo, Slonovo uvo, Mala gamba, Gamba, Amfora and Bela dolga.The virus status of the pepper plants was analyzed on seedlings and on pepper plants throughout the vegetative season.The seedling material was collected in May, while the samples from grown pepper plants were collected three times during the vegetation in 2009 (in the second half of June, in the beginning of August and at the end of September).For the analysis of seedlings whole plants were collected, while for analyzing the grown pepper throughout the vegetation, leaf samples from the upper part of the plants were collected.Leaf samples were collected from the same marked plants.

In
In 2008, plants were tested for the following viruses: CMV, AMV, TMV, PVY and PVX, while in 2009, the samples were tested for the previous five viruses, as well as for TSWV.The collected samples were tested for virus infections using the double antibody sandwich ELISA (DAS -ELISA) as described by Clark and Adams (1977) and modified and proposed by Bioreba (1999).The samples were tested in duplicate and results were recorded on MULTISCAN ASCENT.

RESULTS AND DISCUSSION
The results from the analyzed leaf samples in 2008 are presented in Table 1.Results presented in Table 1, show that in both regions, the most prevalent virus was CMV, which was found in 13 of 14 tested samples (92.8%).In the locality Bp, out of five probed viruses, four were detected (CMV, AMV, TMV and PVY), while in the locality Te only CMV infections were detected.AMV was detected in: Bp1, Bp4 and Bp9 samples.TMV was detected in the samples Bp4 and Bp3, while PVY was found in two samples (Bp5 and Bp7).PVX was not found in any of the analyzed samples.
The results in Table 2 show that only two samples from the seedling material (Sk2 and St2) were infected with PVY.The other samples were virus free.This condition of the seedling material was expected.This indicates that the pepper seeds were healthy and virus free (PVY does not transmit by seed (Šutić, 1995)) and that the virus infections on the plants were generally induced by vectors or by mechanical means throughout the vegetation (Brunt et al., 1996;Büchen-Osmond, 2006).The results from the samples analyzed in 2009 in 7 areas of R. Macedonia are presented in Figure 2. The results given in Figure 2 show that pepper plants cultivated on open fields were susceptible to virus infections in all of the examined areas.
In five of the seven analyzed areas the most widely spread was the infection by CMV, while in the other two examined areas the most prevalent were the infections

Virus
Banski pat (Bp) Teranci (Te) +, the sample is infected with a certain virus; -, the sample is not infected with a certain virus.  1 2 3 4 5 6 1 2 3 1 2 +, the sample is infected with a certain virus; -, the sample is not infected with a certain virus.The results given in Table 1 and Figure 2, are fully corresponding with the results reached by other authors (Bogatzevska et al., 2007).Furthermore, Jovancev et al.   (1996) indicated the regular presence of CMV, AMV, TMV, TSWV and PVY in the pepper production in open fields.The domination of CMV infection in similar pepper production was also established by Choi et al. (2005).
In 2009, samples from grown pepper plants were collected three times during the vegetation (in the second half of June, in the beginning of August and at the end of September).Analyzing the OD absorbance with DAS.
ELISA tests, it is possible to follow the dynamics of the virus infection throughout the vegetation in the tested plants (Rusevski, 2002).In Table 3, the dynamics of the virus infection is demonstrated, while the dynamics of the maximum and the average accumulation of virus antigens are shown in Tables 4 and 5.
Table 3 shows that in the beginning of the vegetation (during the first examination), the number of infected plants per region was small (0-5 infected plants per region).During the second inspection, the number of infected plants in most of the regions has enlarged, for example in sample Ko(b) the number of infected pepper   plants with CMV has grown from 2 up to 17 (15 new infected plants).During the third examination, the dynamics of the plants infection differed depending on the probed virus and region.For example, the number of infected plants with CMV in sample Pr(c), grew from 2 up to 6 (4 new infected plants).In some cases the number of infected plants stayed the same, for example in sample Pr(c) on pepper plants infected with TSWV (0 new infected plants).Using established data, mixed virus infections, in all of the tested regions, were another aim of observation.Usually the mixed infections were consisted of CMV and some other virus, because CMV was the most spread virus.Exception was in Ra, where CMV did not occur in any mixed infection, while mixed infection was detected between AMV and TSWV.

Studied regions and number of repetitions
The laboratory analysis showed curious data, where in one marked plant from Ku(b) a mixed infection with all of the established viruses was detected.Table 4 demonstrates that during the first inspection, most of the probed viruses had not obtained their maximum antigen accumulation in the infected plants.Exceptions represented virus infections with TMV and AMV in some of the analyzed regions (Pr(a) -0.648, Ko(a) -0.524).In most of the virus infections the maximum antigen accumulation was observed in the second examination.The highest maximum of the probed viruses was observed at CMV.In five of the seven examined regions the maximum antigen accumulation for CMV was above 1.The highest maximum was found in sample Si(b) -1.695.None of the other probed viruses obtained maximum antigen accumulation in the pepper plants above 1.During the third inspection, most frequently, the virus antigen accumulation in the infected plants reduced.The virus antigen accumulation of TSWV in the pepper plants in sample Ra(c) declined towards near to negative serological reaction (0.251).Because of the reduction of virus antigen accumulation in the infected plants, the symptoms become less visible, which may be due to the content of the virions in the leaf sap (Brunt et al., 1996;Büchen-Osmond, 2006).In fewer cases, the maximum virus antigen accumulation in the pepper plants was observed in the third inspection, (sample St(c) where the maximum antigen accumulation of CMV was 1.489).
Tables 4 and 5 demonstrate that the dynamics of the maximum virus antigen accumulation and the average virus antigen accumulation, almost completely overlap throughout the tested period.Table 5 shows that the highest average virus antigen accumulation mostly was not found during the first examination of the pepper plants.Exceptions represented virus infections with TMV and AMV in some of the analyzed regions (Pr(a) -0.648, Ko(a) -0.376), as demonstrated previously in Table 4.For most of the probed viruses the highest average antigen accumulation in the infected plants was established in the second inspection.The highest average virus antigen accumulation was attained by CMV.In five of the seven studied regions, the average antigen accumulation for CMV was above 1.The highest average virus antigen accumulation was found in sample Pr(b) with 1.563.None of the other probed viruses obtained average antigen accumulation in the pepper plants above 1.During the third examination, most frequently, the virus antigen accumulation in the infected plants reduced (sample Ku(c) infection with AMV -0.256), as shown previously in Table 4. Laboratory data reached confirmation at the plants symptomatology in situ.Similar to the data above (Table 4) in some cases, the highest average virus antigen accumulation in the pepper plants was observed in the third inspection (PVY-0.664, in sample Ko(c)).The most evident difference between the maximum and the average virus antigen accumulation was observed in the infected plants by CMV in St during the third examination.Thus, average virus antigen accumulation was 0.695 (Table 5), while the maximum virus antigen accumulation observed in one of the infected plants was 1.489 (Table 4).Variation of the dynamics of the virus antigen accumulation (Tables 4 and  5) explains the less visibility of symptoms in the infected plants.The phenomena of acute, chronically and recovery phase in plants infected with viruses (eg.tobacco and pepper plants related to TMV), have been pointed before (Šutić, 1994).Also, according to CMV, AMV and TMV description, the number of virions in a virus preparation can vary and it can contain few or many virions (Brunt et al., 1996;Büchen-Osmond, 2006a, b, c).

Conclusion
Regarding the results presented above, generally it can be concluded that: (1) The pepper seedling material was generally healthy, except for the infections by PVY of the two samples from the areas around Skopje and Strumica.
(2) The pepper plants cultivated in open fields in R. Macedonia were susceptible to various virus infections (CMV, AMV, TMV, PVY and TSWV).
(3) In 2008 and 2009 the most widely spread virus infection on the pepper plants was the infection by CMV.(4) The infections by AMV, TSWV, TMV and PVY were less present on the pepper plants in the examined regions.
(5) PVX infections were not present in the pepper plants in any of the examined regions.(6) For most of the probed viruses during the first inspection, the virus infections were expanding and had not reached their maximum.(7) The maximum virus antigen accumulation and the highest average virus antigen accumulation in the infected plants were usually observed in the second inspection throughout the vegetation.
(8) During the third inspection, the phenomenon of less visible symptoms in infected plants was observed and reduction of virus antigen accumulation in the infected pepper was detected.
2008 and 2009 research was conducted to determine the virus status of the pepper plants cultivated in open fields in R. Macedonia.Pepper plants cultivated in open fields were analyzed, because they are more susceptible to virus infections, compared to pepper cultivated in greenhouses

Figure 1 .
Figure 1.Map of R. Macedonia with the sampling locations.
order to determine the presence of most common viruses of pepper plants grown in the open fields in R. Macedonia, in 2008 Kocani was chosen as a representative region.The collection of virus suspected material (plants with typical symptoms of virus infections) was performed in September 2008, from two localities around the Kocani region: Banski pat [Bp] and Teranci [Te].From the locality Banski pat 9 samples were collected and from Teranci 5 samples were collected.All samples from this region derived from variety Kurtovska kapija.In 2009 the samples were collected from the most important regions for pepper production in R. Macedonia: around Skopje [Sk], Kumanovo [Ku], Kocani [Ko] (Banski pat [Bp] and Teranci [Te]), Strumica [St] (Prosenikovo [Pr] and Robovo [Ro]), Radovish [Ra] (Injevo [In] and Oraovica [Or]), Prilep [Pr] (Malo Konjari [Ma]) and municipality Rosoman (Sirkovo [Si]) (Figure 1).

Figure 2 .
Figure 2. Virus status of pepper in 7 areas in R. Macedonia in 2009 (% of infection).
Dynamics of virus infection on pepper plants throughout the vegetation in 2009.
locations per region are examined, 7 plants per location, 2 2 locations per region are examined, 7 plants per location, 3 3 locations per region are examined, 7 plants per location, a, b, c different sampling periods (asecond half of June; bbeginning of August; cend of September).Table 4. Dynamics of maximum virus antigen accumulation in individual plants (according OD absorbance) throughout the vegetation on pepper plants in 2009.

Table 1 .
Results from DAS ELISA on pepper specimens (variety Kurtovska kapija) in 2008 in the area around Kocani.

Table 2 .
Results from DAS-ELISA on specimens of pepper seedlings in 2009.

Table 5 .
Dynamics of average virus antigen accumulation (according OD absorbance) throughout the vegetation on pepper plants in 2009.
4 locations per region are examined, 7 plants per location, 2 2 locations per region are examined, 7 plants per location, 3 3 locations per region are examined, 7 plants per location, a, b, c different sampling periods (asecond half of June; bbeginning of August; cend of September), -, the sample is not infected with a certain virus. 1